Targeted disruption of HoxD regulation during limb formation

在肢体形成过程中有针对性地破坏 HoxD 调节

基本信息

项目摘要

The limbs of tetrapod vertebrates are composed of four basic segments: the limb, forelimb, wrist/ankle, and hand/foot. The identity of these segments is determined during limb formation by specific combinations of Hox genes. As the limb bud grows away from the main body axis, two waves of Hoxd gene expression occur. The first wave specifies the limb and forelimb. Then, following a short period of low Hoxd expression, a second wave of expression specifies the hand and foot portions. Cells that are produced during the transient step between these waves will form the wrist and ankle. Remarkably, these two phases of expression result from topological changes within the HoxD gene cluster and flanking regulatory domains. During formation of the arm and forearm, enhancers in the telomeric regulatory domain (T-DOM) drive the first collinear wave of expression. Then in proliferating distal limb bud cells, the T-DOM is inactivated, followed shortly by activation of the centromeric regulatory domain enhancers (C-DOM), driving the second wave of Hoxd expression and formation of the hand. The HoxA transcription factor HOXA13 is essential to this transition, acting to repress T-DOM and then activating C-DOM. The goal of the work proposed here is to understand how changes in enhancer status drive the topological transition between the two waves of Hoxd expression. To do this, we will perform two key experiments to challenge the conversion of each regulatory domain from active to inactive, or vice-versa. Specifically, we will first disrupt the activation of C-DOM by deleting an important autopod (hand/foot) enhancer called II-1. Our preliminary data indicates that HOXA13 acts through II-1 to drive the second wave of Hoxd expression. We will then target a copy of this enhancer into T-DOM, providing HOXA13 with an enhancer element that it normally acts through to activate C-DOM, but now within the chromatin context of T-DOM, which is being silenced in the same cells. We will evaluate changes in the chromatin state induced by these genetic perturbations by monitoring chromatin modifications via histone ChIP-Seq (H3K27me3, H3K4me1, and H3K27Ac) and ATAC-Seq. We will monitor for changes in gene expression quantity by RNA-Seq, and changes in the location of expression by whole-mount in situ hybridization. Furthermore, because we expect these mutations to induce large-scale changes in chromatin topology and possibly force continued activity from native T-DOM enhancers, we will utilize Capture Hi-C to monitor changes in HoxD topology. One prediction from these experiments is that each modified regulatory domain will resist complete conversion from one state to the other, due to the influence of this enhancer, with significant changes in chromosomal topology and gene expression. This experimental approach mirrors some natural mutations in chromatin structure that influence human development and health. Together these experiments will shed light on the molecular mechanisms of limb patterning, developmental gene regulation, and the influence of enhancers on genome topology.
四足脊椎动物的四肢由四个基本部分组成:四肢,前肢,腕/踝, 手/脚这些节的身份是在肢体形成过程中通过以下特定组合来确定的: Hox基因。当肢芽远离主体轴生长时,Hoxd基因的表达出现两波。 第一个波指定肢体和前肢。然后,在短时间的低Hoxd表达后, 第二波表情指定手部和脚部。在短暂的时间内产生的细胞 这些波浪之间的一步将形成手腕和脚踝。值得注意的是,这两个阶段的表达结果 HoxD基因簇和侧翼调控结构域的拓扑变化。形成期间 在手臂和前臂,端粒调节结构域(T-DOM)中的增强子驱动第一个共线波, 表情然后在增殖的远端肢芽细胞中,T-DOM失活,随后很快被激活 着丝粒调控域增强子(C-DOM),驱动Hoxd表达的第二波, 手的形成。HoxA转录因子HOXA 13对这种转变至关重要,其作用是抑制 T-DOM,然后激活C-DOM。这里提出的工作目标是了解 增强子状态驱动Hoxd表达的两个波之间的拓扑转变。为此,我们将 进行两个关键实验,以挑战每个调节结构域从活性到非活性的转换,或 反之亦然。具体来说,我们将首先通过删除一个重要的autopod来破坏C-DOM的激活 (hand/英尺)增强子称为II-1。我们的初步数据表明,HOXA 13通过II-1来驱动 第二波Hoxd表达。然后,我们将这个增强子的一个拷贝定向到T-DOM中,提供HOXA 13 它通常通过一个增强子元件来激活C-DOM,但现在在染色质内, T-DOM的上下文,它在相同的单元格中被沉默。我们将评估染色质状态的变化 通过组蛋白ChIP-Seq监测染色质修饰, (H3 K27 me 3、H3 K4 me 1和H3 K27 Ac)和ATAC-Seq.我们将监测基因表达的变化 通过RNA-Seq定量,通过整体原位杂交检测表达位置的变化。 此外,因为我们预期这些突变会诱导染色质拓扑结构的大规模变化, 可能迫使天然T-DOM增强子继续活动,我们将利用Capture Hi-C监测 HoxD拓扑结构的变化。这些实验的一个预测是,每个修饰的调控结构域将 抵抗从一种状态到另一种状态的完全转换,由于这种增强剂的影响, 染色体拓扑结构和基因表达的变化。这种实验方法反映了一些自然的 染色质结构的突变影响人类发育和健康。这些实验 将阐明肢体模式的分子机制,发育基因调控, 增强子对基因组拓扑结构的影响。

项目成果

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